THE PML-RAR ONCOGENIC FUSION PROTEIN AND ITS ROLE IN ACUTE PROMYELOCYTIC LEUKEMI

这个子项目是许多利用 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 急性早幼粒细胞白血病（APL）是由15号和17号染色体之间的特异性相互易位引起的。这种易位融合了PML肿瘤抑制基因和视黄酸受体a（RARa）基因，产生了PML-RARa嵌合癌蛋白，干扰了与骨髓分化相关的信号传导。APL患者接受全反式视黄酸（ATRA）治疗，但由于融合蛋白的RAR α部分的配体结合结构域（LBD）中赋予ATRA抗性的突变而总是经历复发。这些突变影响蛋白结合视黄酸的能力，以及分别经历核辅阻遏物（N-CoR）和辅激活物（ACTR）蛋白的释放和结合。这些辅因子在RAR α转录功能中发挥重要作用。我们开发了荧光各向异性和共振能量转移测定，以定量结合的辅激活子和辅阻遏肽的野生型RARa LBD和四个ATRA耐药突变体在不同浓度的ATRA的存在下。通过使用这些试验，我们已经确定了辅因子的解离常数，并确定了在这些患者和细胞系突变的辅因子招募方面的ATRA耐药的基础。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Acute Promyelocytic Leukemia (APL) results from a specific reciprocal translocation between chromosomes 15 and 17. The translocation fuses the genes for the PML tumor suppressor and retinoic acid receptor a (RARa), generating a PML-RARa chimeric oncoprotein that interferes with signaling associated with myeloid differentiation. APL patients receive treatment with all-trans retinoic acid (ATRA), but invariably undergo relapse owing to mutations imparting ATRA resistance in the ligand binding domain (LBD) of the RARa portion of the fusion protein. The mutations affect the ability of the protein to bind retinoic acid, as well as undergo release and binding of nuclear co-repressor (N-CoR) and co-activator (ACTR) proteins, respectively. These cofactors play essential roles in RARa transcription function. We developed fluorescence anisotropy and resonance energy transfer assays to quantitate the binding of coactivator and corepressor peptides to the wild-type RARa LBD and four ATRA resistant mutants in the presence of varying concentrations of ATRA. By use of these assays, we have determined cofactor dissociation constants and defined the basis of ATRA resistance in these patient and cell line mutations in terms of cofactor recruitment.